Human plasma serves as a source for deriving valuable proteins. Therapeutic proteins derived from human plasma have been used in the treatment of a broad range of diseases including primary immune deficiencies (immunoglobulin G), critical care involving hypovolemia (albumin), wound healing (fibrinogen), and hereditary deficiencies such as hemophilia A (Factor VIII), hemophilia B (Factor IX), von Willebrand's disease (vWF), and congenital emphysema resulting from a deficiency of alpha-1 Proteinase Inhibitor (A1PI). Besides its application as a raw material for isolating very valuable proteins, whole human plasma continues to be major source of coagulation factor replacement therapy for patients with clotting factor deficiency. Human plasma and therapeutic proteins derived therefrom are used to treat about more than one million patients globally each year. The global demand of such therapeutics is significantly greater than the current level of supply.
For patients in the need of whole plasma for therapeutic purposes, it is available as either fresh frozen plasma or liquid plasma. Fresh frozen plasma (FFP) is the plasma removed from a unit of whole blood and frozen at or below −18° C. within eight hours of blood collection as a single-donor plasma unit. Liquid plasma is stored at temperatures of 4-8° C. within 4 hours of blood collection and separated from the red blood within 48 hours of blood collection. Each of these plasma units are from single donors and individually tested for viral markers and, in regards to viral transmittance, single donors are considered to be reasonably safe. However, there continues to be a small but defined risk of viral transmission, because such plasma units usually do not undergo a process of viral inactivation to kill viruses like HIV, hepatitis B, hepatitis C, and other viruses which could potentially cause disease.
For the purpose of deriving the therapeutic proteins, a large number of fresh frozen plasma units are pooled together from various donors. Human plasma proteins for therapeutic use have been manufactured from large pools of plasma for over 50 years. One of the important concerns of single donor or pooled plasma, however, is viral safety. Though every donor who contributes to the pool of plasma is tested individually for viruses, including HIV, HBV, HCV, etc. before blood or plasma donation, there remains a small risk of infection with viruses due to “window period donations,” that is donations made between the initial acquisition of infection and the detection of a positive test result with existing diagnostics due to inherent technical limitations. Even a single donor infected with a pathogen, which remained undetected after screening, can potentially contaminate an entire pool of plasma and infect many or all recipients exposed to the pool. Therefore, there is a need to address the viral safety of pooled plasma or the therapeutic proteins derived therefrom.
To render plasma or plasma-derived therapeutic proteins virus-safe, various methods have been attempted to remove or inactivate viruses. For virus inactivation, biological fluids of interest are subjected to physical treatments like pasteurization, wherein the pooled plasma is subjected to wet heat at a temperature of about 60° C. for periods of about 10 hours, or treated with dry heat during which the product of interest is treated at higher temperature of about 80° C. for prolonged periods as long as about 72 hours. Such treatments often are found to damage, denature, or vary valuable protein factors, especially labile blood-coagulating components under conditions to which the biological samples are subjected for inactivating the viruses efficiently. During such inactivation processes, the labile coagulation components of the mammalian blood plasma may get inactivated or denatured as much as up to the extent of 50-90% or more present in the untreated plasma. The coagulating components which may be lost during such treatment include valuable plasma factors like factors II, VII, IX, X; plasma fibrinogen (factor I), IgM, hemoglobin, interferon, etc. Therefore, attempts have been made to incorporate steps suitable for protecting proteins of interest.
Other methods for viral inactivation involve treatment with β-propiolactone, formaldehyde, sodium hypochlorite, and the like. However, these methods are generally not considered to be very safe. These methods not only tend to denature the valuable protein components, but also pose difficulties in complete removal of agents such as β-propiolactone which is deleterious and has shown to be carcinogenic in animals and is dangerous even to personnel handling it.
One of the most commonly employed methods for viral inactivation of plasma or plasma derived protein products is solvent detergent treatment. Solvent detergent treated plasma has been approved for use in the treatment of patients with documented deficiencies of coagulation factors for which there are no concentrate preparations available, including congenital single-factor deficiencies of factors I, V, VII, XI and XIII, and acquired multiple coagulation factor deficiencies; reversals of warfarin effect; and treatment of patients with thrombotic thrombocytopenic purpura (TTP). A cost-effectiveness analysis for solvent-detergent-treated frozen plasma (SDFP), calculated a cost of $289,300 per quality-adjusted life year (QALY) saved. (Jackson et al. JAMA.1999; 282: 329). Solvent-detergent treatment is particularly effective for enveloped viruses such as Vesicular Stomatitis Virus (VSV), Pseudorabiesvirus (PRV), Semliki Forest Virus (SFV) and Bovine Diarrhoea Virus (BVDV). (Seitz et al. Biologicals, 30(3): 197-205(9) (2002)). Solvent detergent treatment is primarily employed to reduce the already-low risk of viral transmitted fresh frozen plasma from donors in the infectious, seronegative window period of currently known viral infections and the risk of transmission of lipid enveloped viruses not currently recognized as a risk to transfusion safety may very well could still be a potential risk in the future.
In a solvent detergent treatment for virus inactivation, the protein-containing composition is contacted with dialkyl- or trialkylphosphate, preferably with mixtures of trialkylphosphate, and detergent, usually followed by removal of the dialkyl- or trialkylphosphate (see U.S. Pat. No. 4,540,573). The '573 patent employed dialkyl- or trialkylphosphate in an amount between about 0.01 mg/ml and about 100 mg/l. The amount of detergent employed, according to the '573 patent, could range from about 0.001% to about 10%. Similarly, U.S. Pat. No. 4,314,997 employed a detergent concentration could vary from 0.25% to as high as about 10%.
Another detergent approach of viral inactivation is to subjecting plasma protein products to prolonged contact with non-denaturing amphiphile (see U.S. Pat. No. 4,314,997). Amphiphiles can be anionic, cationic, nonionic detergents. The amphiphilic detergent molecule is hydrophobic at one end and hydrophilic at the other end, which makes it useful for purification of therapeutic blood proteins. Ionic detergents, either anionic or cationic tend to more active than nonionic detergents. While being effective at destroying viruses, detergents may readily destroy or damage living cells. Detergents are capable not only of destroying viruses, but also disrupting other vital lipid-based structure like biomembranes that surrounds and form a significant internal structural component of every animal and plant cell. Further, high concentrations of detergent are likely to damage or denature proteins that are present and/or desirable for isolation from the biological sample. The incubation of plasma, plasma derived therapeutic proteins, plasma cryoprecipitate, or plasma cryosupernatants with such high concentration of the detergents not only would harm the plasma components but also are known to damage biomembranes. Moreover, the high concentration of detergent is extremely harmful when injected intravenously and hence such detergent-treated plasma would not be suitable for injections. To avoid damage to living cells and proteins, a lower concentration of detergent can be employed, but at the risk of being ineffective for viral inactivation. Therefore, to ensure living cells and proteins are not damaged and at the same time viral inactivation is effective, removal of detergents is imperative.
Commonly employed methods for removal of detergent include affinity or ion-exchange chromatography. These methods are lengthy and time consuming, and involve multiple steps. As one skilled in the art would appreciate, each recovery step is often associated with the loss of proteins of interest and hence result in lower yield. Further, these methods are suitable only for the particular protein factor as an end product and would not be appropriate for the whole plasma. To make it applicable to whole plasma, whole plasma would need to be reconstituted after each of the factors is successively separated and purified. After each step, some amount of time and product would be lost, which may ultimately lead to significant overall loss.
After solvent-detergent treatment, the detergent can be removed by employing several steps chosen among diafiltration, adsorption on chromatographic or affinity chromatographic supports, precipitation and lyophilization, etc. Dialkyl- or trialkylphosphate is often removed by precipitation of the protein with glycine and sodium chloride (see U.S. Pat. No. 4,540,573). The process of the '573 patent is particularly time-consuming as nonionic detergents employed with the trialkylphosphate are removed by diafiltration using either insolubilization or lyophilization. One skilled in the art appreciates that these processes are cumbersome, expensive, time consuming, and/or can result in loss of vital components of plasma.
Removal of detergent and solvent can additionally be preformed by partitioning the protein solution against an organic liquid such as castor or soy bean oil. The detergent and solvent partition into the organic liquid and are thus eliminated. The organic liquid that is oil is then removed by chromatography. This procedure involves partitioning of plasma and regenerating or replacing the chromatographic components, which tend to be very tedious, time consuming and cost intensive.
Another method for reducing of virus-inactivating chemicals and/or detergent is by high salting out effect (see U.S. Pat. No. 5,817,765). In this procedure, a concentration above 0.5M of salt with high salting out effect according to Hofmeister series is added to the aqueous plasma protein solution for forming vesicles containing the virus-inactivating chemicals and/or detergents. The vesicles are removed from the aqueous phase, for example by phase separation or filtration. The technique of phase separation, however, particularly that of vesicles, is laborious, imprecise, and difficult method, which would be very cumbersome in large scale operations because of operational issues like cleaning, sanitization, process validation, etc. Additionally, the further step of protein recovery from aqueous solution may result in the loss of final protein yield. Any trace of salt remaining in the plasma product, would also be undesirable as it would render it unsuitable for the most therapeutic applications.
Removal of solvent/detergent can alternatively be performed by using carbon either in the form of activated carbon or charcoal. For example, Chinese Patent number CN 1371992 employs solid phase material containing active carbon as adsorbent for removing organic solvent used as virus inactivator and/or detergent from aqueous solution. U.S. Pat. No. 5,834,420 uses precipitation of viral inactivated fraction in a solution containing an amino acid at an acidic pH and filtering. Preferably the filtration step is carried out through a filter of activated carbon of which AKS-4 and AKS-7 are particularly suited. Carbon, however, is a nonspecific adsorbent, and when employed to adsorb virus inactivator and/or detergent, may also adsorb some of the important peptides components of interest from the plasma. The use of carbon could result in a final product devoid of these useful components.
U.S. Pat. No. 6,610,316 discloses a “sugar detergent” rendered insoluble by being bound to an inert substrate. The method described in the '316 patent requires an additional step of binding the detergent to the resin. Further, additional testing protocol could be required to check or ensure sufficient binding of the detergent in order to avoid leaching of the detergent into the blood solution. Detergent that is not bound sufficiently to the resin could contaminate the blood product and render it unsafe for the desired use. Further, the method disclosed in the '316 patent is directed to blood or aqueous liquid containing blood cells, and does not demonstrate the suitability of this method for plasma or plasma derived proteins.
From the forgoing reasons, it is evident that it is imperative to treat plasma or plasma derivatives with virus-inactivating agents to render it safe for therapeutic applications. It is also important to improve such virus-safe plasma or plasma derivatives by clearing the virucidal agents employed for inactivating viruses to desired acceptable level for clinical use. However in view of the drawbacks associated with the methods discussed above, there continues to exist a need for providing a simple, reproducible process, not compromising time consumed and yield, yet easily validated for improving virus-safe biological fluids including plasma or plasma derivatives by clearing virucidal agents like detergent and solvent to acceptable levels using effective and simple methods without significantly affecting the plasma composition.